Acetohydroxyacid synthase (AHAS; EC 4.1.3.18), also known as acetolactate synthase (ALS), is the first enzyme that catalyzes the biochemical synthesis of the branched-chain amino acids valine, leucine, and isoleucine (Singh B. K. (Ed) Plant amino acids. Marcel Dekker Inc. New York, N.Y. Pg 227-247). AHAS is the site of action of four structurally diverse herbicide families including the sulfonylureas (LaRossa R A and Falco S C, 1984 Trends Biotechnol. 2:158-161), the imidazolinones (Shaner et al., 1984 Plant Physiol. 76:545-546), the triazolopyrimidines (Subramanian and Gerwick, 1989, Inhibition of acetolactate synthase by triazolopyrimidines in (ed) Whitaker J R, Sonnet P E Biocatalysis in agricultural biotechnology. ACS Symposium Series, American Chemical Society. Washington, D.C. Pg 277-288), and the pyrimidinylbenzoates (Subramanian et al., 1990 Plant Physiol 94: 239-244.). By inhibiting AHAS activity, these families of herbicides prevent further growth and development of susceptible plants, including many weed species. Imidazolinone (IMI) and sulfonylurea (SU) herbicides are widely used in modern agriculture due to their effectiveness at very low application rates and relative non-toxicity in animals.
In plants, the AHAS enzyme is comprised of two subunits: a large subunit (catalytic role) and a small subunit (regulatory role) (Duggleby and Pang (2000) J. Biochem. Mol. Biol. 33:1-36). The AHAS large subunit (AHASL) can be encoded by a single gene, as in the case of Arabidopsis and rice, or by multiple gene family members, as in maize, canola, and cotton. Specific, single-nucleotide substitutions in the large subunit can confer upon the enzyme a degree of insensitivity to one or more classes of herbicides (Chang and Duggleby (1998) Biochem J. 333:765-777). By convention, modifications of the AHAS amino acid sequence are typically identified in reference to their position in the Arabidopsis thaliana AHAS sequence (EMBL Accession No. X51514) and denoted with (At).
Modifications of AHAS genes can result in herbicide tolerant phenotypes (Hattori, 1995; Warwick, 2008). Substitutions in genes encoding the AHAS large subunit, which are referred to herein as AHASL genes, are the molecular basis of herbicide tolerance in CLEARFIELD® crops, which have increased tolerance to imidazolinone herbicides. Because each of these substitutions results in a semi-dominant phenotype, one substitution in a heterozygous state may be sufficient to produce a level of herbicide tolerance that is acceptable for many crop productions systems. However, for particular herbicide applications, and in cases with crop plants having multiple AHASL genes such as wheat, combinations of substitutions are desired to achieve an increased level of tolerance to herbicides.
Triticum aestivum is a hexaploid wheat species having three genomes, i.e., termed a D genome, a B genome, and an A genome. Each genome contains an AHAS gene and the genes have been named to take into account genome of origin and evolutionary relatedness, for example the AHAS large subunit gene located on the D, B, and A genomes of Triticum aestivum are referred to as TaAHASL1D, TaAHASL1B, and TaAHASL1A, respectively.
Each of the genes exhibit significant expression based on herbicide response and biochemical data from variants in each of the three genes (Ascenzi et al. (2003) International Society of Plant Molecular Biologists Congress, Barcelona, Spain, Ref. No. S10-17). The coding sequences of all three genes share extensive homology at the nucleotide level (WO 03/014357). Through sequencing the AHASL genes from several varieties of Triticum aestivum, the molecular basis of herbicide tolerance in most imidazolinone (IMI)-tolerant lines was found to be a single base pair change that results in the amino acid substitution S653(At)N (S653N), indicating a serine to asparagine substitution at a position equivalent to the serine at amino acid 653 in Arabidopsis thaliana (WO 03/014356; WO 03/014357). The S653N substitution is due to a single nucleotide polymorphism (SNP) in the DNA sequence encoding the AHASL protein. The substitution has been identified in all three genomes and must be easily and quickly distinguished for accurate breeding and confirmation of commercial seed lots (Berard, 2009; Dong, 2009).
One goal of plant breeders is to introduce imidazolinone tolerance into existing wheat lines by inducing the S653N substitution in the existing lines or by crossing non-IMI-tolerant lines with IMI-tolerant lines followed by backcrossing and selection for imidazolinone tolerance. Another goal of plant breeders is to produce wheat plants with increased levels of imidazolinone tolerance, beyond the levels of tolerance seen in wheat plants possessing a single S653N substitution in a single wheat AHASL gene. Thus, it is desirable to breed wheat plants that possess combinations of S653N substitutions at two or more of the AHASL genes. In addition, it is also desirable to breed wheat plants that are homozygous for the substituted S653N allele at one or more of the AHASL genes. However, to develop the desired wheat plants, rapid methods for identifying the desired plants are needed. Existing methods of detecting wheat plants with imidazolinone tolerance are not highly efficient for use in the development of plants that possess more than a single S653N allele at a single AHASL gene.
Existing methods of identifying plants with enhanced imidazolinone tolerance include field or greenhouse herbicide spray tests and biochemical assays for AHAS activity. However, such methods are time consuming and generally not suited for distinguishing, among large numbers of individual plants, subtle increases in imidazolinone tolerance that may occur when a second S653N allele is introduced into a wheat plant.
Alternative methods for identifying desired plants include DNA-based methods. For example, the AHASL genes, or portions thereof, can be amplified from genomic DNA by polymerase chain reaction (PCR) methods and the resulting amplified AHSL gene or portion thereof can be sequenced to identify the substituted S653N allele and the particular AHASL gene that it is present in. However, such a DNA-sequencing-based method is not efficient for large numbers of samples. Another approach involves that use of radiolabelled or non-isotopically tagged, allele-specific oligonucleotides (ASOs) as probes for dot blots of genomic DNA or polymerase chain reaction (PCR) amplified DNA (Connor et al. (1983) Proc. Natl. Acad. Sci. USA 80:278-282; Orkin et al. (1983) J. Clin. Invest. 71:775-779; Brun et al. (1988) Nucl. Acids Res. 16:352; and Bugawan et al. (1988) Biotechnology 6:943-947. While such an approach is useful for distinguishing between two alleles at a single locus, this approach is not useful for the wheat AHASL genes, because three AHASL genes are nearly identical in the region surrounding the SNP that gives rise to the substituted S653(At)N AHASL protein. Thus, a set of six such oligonucleotide probes could not be developed that would be able to distinguish between the substituted and wild-type alleles at each of the three wheat AHASL genes.
One method that can be adapted for rapidly screening large numbers of individuals for the analysis of an SNP is the amplification refractory mutation system (ARMS) (Newton et al. (1989) Nucl. Acids Res. 17:2503-2516). This PCR-based method can be used to distinguish two alleles of a gene that differ by a single nucleotide and can also be used to distinguish heterozygotes from homozygotes for either allele by inspection of the PCR products after agarose gel electrophoresis and ethidium-bromide staining. The ARMS method is based on the premise that oligonucleotides with a mismatched 3′-residue will not function as primers in PCR under the appropriate conditions (Newton et al. (1989) Nucl. Acids Res. 17:2503-2516). An amplification-refractory mutation system (ARMS-PCR) using agarose-based gel detection methods may incorporate an internal nucleotide mismatch within an allele-specific (AS) primer to enhance the specificity of the assay. For example, the position of an internal mismatch was investigated in a multiple allelic system in a chicken population where the optimum primer had a mismatch 2 base pairs from the polymorphic nucleotide.
The usage of deliberate mismatches in quantitative PCR (qPCR) has been tested in the medical field. Single nucleotide polymorphism (SNP) genotyping is commonly performed to assess disease susceptibility and chimerism assessment. Development of one qPCR system has been attempted for monitoring chimerism. The SNP-specific qPCR assay was able to detect the positive template allele at 0.1%. The AS primers contained one to two intentional mismatches within 1-4 base pairs of the polymorphism of interest.
Mismatches in primers targeting a polymorphic region of a gene in a non-polyploid organism have been found to create a shift in the CT towards higher values during qPCR with SYBR-green detection (for example, the lim1 gene of Picea glauca.) This reduces assay sensitivity making this technique inadequate for pooled samples. The shift is less dramatic when the mismatch is located closer to the 3′ end of the primer. However, it is necessary to decrease the DNA concentration in order to avoid voluntary addition of mismatches.
While ARMS-PCR has proven useful for the analysis of a SNP at a single gene, whether this method, or any other PCR-based methods, can be used be used for the analysis of SNPs that gives rise to herbicide-tolerant substitutions in native AHAS genes in different genomes of the same species, for example, the S653N substitution in each of the three wheat AHASL genes has not been reported. Existing methods experience a lack of assay sensitivity which can make them undesirable for SNP detection, particularly for SNP detection in and analysis of highly homologous genes in the same species, for example, in species having multiple genomes.
Thus, there remains a need for a method which can more efficiently distinguish between wheat plants having wild-type AHASLs and wheat plants having variant AHASLs and the zygosity of the same.